Disulfide Stabilised Antibodies and Fragments Thereof

ABSTRACT

The present disclosure provides an antibody or antibody fragment comprising at least one Fab molecule, wherein the light chain variable region, V L  and the heavy chain region, V H  of the Fab molecule are linked by one or more disulfide bonds, and use of the same in treatment or prophylaxis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/637,209, filed Dec. 17, 2012, issued as U.S. Pat. No. 9,045,529 onJun. 2, 2015, which is a U.S. National Phase of InternationalApplication No. PCT/GB2011/050608 filed Mar. 22, 20111, which claimspriority from GB Patent Application No. 1005064.9 filed Mar. 25, 2010.The entire disclosure contents of these applications are herebyincorporated by reference into the present application.

The present disclosure relates to antibodies and fragments thereof,wherein the variable regions therein are stabilised by an interchaindisulfide bond there between, use of the same in therapy, in particulartherapy by inhalation and pharmaceutical compositions comprising thesame.

Fab and Fab′ molecules are well known in the field of biotherapeuticsand a number formats have been proposed to address various problems. Inessence they are an “antibody” where the constant region —CH₂CH₃ in theheavy chain is absent. The “prime” element refers to the presence of ahinge portion. The present inventors believe that it has never beenproposed to prepare a Fab or Fab′ characterized in that a disulfidebridge is present between the variable regions of a heavy and lightchain pairing therein.

The present inventors believe that a disulfide bond between the variableregions stabilizes the Fab/Fab′ making the final entity more robust. Theimplications are wide ranging in that it may result in a longer shelffor the formulations comprising the modified Fab/Fab′. Additionally itallows new kinds of formulations of the Fab/Fab′ to be prepared, forexample formulations for topical delivery to the lungs, such asformulations for nebulization.

Topic delivery to the lungs is an important area of therapy because itallows rapid and direct delivery to the target organ, whilstsimultaneously minimizing undesirable side effects. Some of the mostuseful and successful therapies are delivered topically to the lungs. Inaddition disease in the lungs and respiratory illness constitutes asignificant burden to health authorities. In 2007 more than 160,390people were expected to die from lung cancer in the US alone and in 2006more than 39,000 cases of lung cancer were diagnosed in the UK.Similarly asthma is a significant problem with an estimated 100 millionsuffers worldwide. Other respiratory disease include COPD, emphysema,and chronic bronchitis. In 2000 the World Health organization estimatedthat 2.74 million people died of COPD worldwide.

Clearly effective treatment of these patients represents a significantchallenge and anything that can be done to facilitate their treatmentwould be very useful. To date very few biotherapies have been availablefor respiratory treatment. Generally antibodies and antibody fragmentsare administered by infusion. The present invention aims to push theboundaries of respiratory medicine further by providing biologicalmolecules which are suitable for use in respiratory therapy.

Thus the present inventors provide an antibody or antibody fragmentcomprising a Fab molecule having a light and a heavy chain, wherein thelight chain variable region, V_(L) and the heavy chain variable region,V_(H), are linked by one or more disulfide bonds.

The disulfide bond between the V_(H) and V_(L) pair seems to aid generalstability of the molecules, for example it aids the chemical and/orphysical stability of the molecule. The presence of the disulfidebond(s) may result in a higher Tm, when the molecules are analysed by asuitable technique such as Thermofluor or Differential Scanningcalorimetry (DSC), than the Tm observed in corresponding molecules wherethe disulfide bond is absent. An increased chemical stability may alsoresult in an increased thermal stability, for example the molecule withdisulfide bonds presents may denature at a higher temperature than amolecule where the disulfide bonds are absent. The disulfide bondbetween V_(H) and V_(L) may also minimize inappropriate aggregation, forexample when the molecule is formulated.

Antibody Fab fragments of the present invention may be any heavy chainand light chain pair having a variable (V_(H)/V_(L)) and constant region(C_(H)/C_(L)).

In one embodiment the format of the present invention consists of a Fabfragment.

In one embodiment the format of the present invention consists of a Fab′fragment.

In one embodiment the format of the present invention consists of adiFab fragment.

In one embodiment the heavy and light chain pair V_(H)/C_(H1) andV_(L)/C_(L) is covalently linked through interchain cysteines in theheavy and light chain constant regions.

The variable domains are provided in each chain such that they formpre-defined pairs with suitable/adequate binding to a target antigen.

In one embodiment, a Fab molecule according to the present invention hasone, two or three, such as one disulfide bond between the V_(H) andV_(L) pair or each pair.

It will be appreciated that a Fab molecule as described herein may formpart of another antibody molecule or antibody fragment. For example theheavy chain of the Fab fragment may be extended by one or more aminoacids to create a Fab′ fragment.

In one example, the present invention provides a Fab′ fragment having aheavy and light chain pair, wherein the heavy and light chain variableregions of the heavy and light chain pair are linked by a disulphidebond.

In one example the present invention provides a complete antibodymolecule comprising two Fab molecules wherein at least one of the Fabmolecules is a Fab molecule according to the present invention. In oneexample the present invention provides a complete antibody moleculecomprising two Fab molecules wherein both Fab molecules are Fabmolecules according to the present invention. i.e. in one Example thepresent invention provides a complete antibody molecule having two heavyand light chain pairs wherein the heavy and light chain variable regionsof each heavy and light chain pair are linked by a disulphide bond. Inone example only one of the heavy and light chain variable region pairsare linked by a disulphide bond.

In one example the present invention provides a F(ab)₂ fragmentcomprising two Fab molecules wherein at least one of the Fab moleculesis a Fab molecule according to the present invention. In one example thepresent invention provides a F(ab)₂ fragment comprising two Fabmolecules wherein both of the Fab molecules are Fab molecules accordingto the present invention. i.e in one Example the present inventionprovides a F(ab)₂ having two heavy and light chain pairs wherein theheavy and light chain variable regions of each heavy and light chainpair are linked by a disulphide bond. In one example only one of theheavy and light chain variable region pairs are linked by a disulphidebond.

Suitable variable domains pairs may be identified by any means possible,for example including generation of antibodies in hosts and screening ofB cells. Alternatively suitable pairs may be identified by phagedisplay. In one embodiment the variable domain pair has affinity for atarget antigen of 100 nM or less, such as 50 nM or less, in particular 1nM or less.

The antibody molecules and variable domain pairs of use in the presentinvention can be from any species but are preferably derived from amonoclonal antibody, a human antibody, or are humanised fragments. Anantibody fragment for use in the present invention can be derived fromany class (e.g. IgG, IgE, IgM, IgD or IgA) or subclass of immunoglobulinmolecule and may be obtained from any species including for examplemouse, rat, shark, rabbit, pig, hamster, camel, llama, goat or human.

In one embodiment, the antibody or antibody fragment e.g. Fab or Fab′ isa monoclonal, fully human, humanized or chimeric antibody fragment. Inone embodiment the antibody or antibody fragment e.g. Fab or Fab′fragments are fully human or humanised.

Monoclonal antibodies may be prepared by any method known in the artsuch as the hybridoma technique (Kohler & Milstein, Nature, 1975, 256,495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., Immunology Today, 1983, 4, 72) and the EBV-hybridomatechnique (Cole et al., “Monoclonal Antibodies and Cancer Therapy”, pp.77-96, Alan R. Liss, Inc., 1985).

Antibodies for use in the invention may also be generated using singlelymphocyte antibody methods by cloning and expressing immunoglobulinvariable region cDNAs generated from single lymphocytes selected for theproduction of specific antibodies by, for example, the methods describedby Babcook, J. et al., Proc. Natl. Acad. Sci. USA, 1996, 93(15),7843-7848, WO 92/02551, WO2004/051268 and WO2004/106377.

Humanized antibodies are antibody molecules from non-human specieshaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework region from a human immunoglobulinmolecule (see, for example, U.S. Pat. No. 5,585,089).

The antibodies for use in the present invention can also be generatedusing various phage display methods known in the art and include thosedisclosed by Brinkman et al., J. Immunol. Methods, 1995, 182, 41-50;Ames et al., J. Immunol. Methods, 1995, 184, 177-186; Kettleborough etal. Eur. J. Immunol., 1994, 24, 952-958; Persic et al., Gene, 1997 187,9-18; and Burton et al., Advances in Immunology, 1994, 57, 191-280; WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; and WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743; and 5,969,108.Also, transgenic mice, or other organisms, including other mammals, maybe used to generate humanized antibodies.

Fully human antibodies are those antibodies in which the variableregions and the constant regions (where present) of both the heavy andthe light chains are all of human origin, or substantially identical tosequences of human origin, not necessarily from the same antibody.Examples of fully human antibodies may include antibodies produced forexample by the phage display methods described above and antibodiesproduced by mice in which the murine immunoglobulin variable and/orconstant region genes have been replaced by their human counterparts eg.as described in general terms in EP0546073 B1, U.S. Pat. No. 5,545,806,U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,625,126, U.S. Pat. No.5,633,425, U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,770,429, EP 0438474B1 and EP0463151 B1.

The antibody Fab or Fab′ fragment starting material for use in thepresent invention may be obtained from any whole antibody, especially awhole monoclonal antibody, using any suitable enzymatic cleavage and/ordigestion techniques, for example by treatment with pepsin.Alternatively, or in addition the antibody starting material may beprepared by the use of recombinant DNA techniques involving themanipulation and re-expression of DNA encoding antibody variable and/orconstant regions. Standard molecular biology techniques may be used tomodify, add or delete amino acids or domains as desired. Any alterationsto the variable or constant regions are still encompassed by the terms‘variable’ and ‘constant’ regions as used herein.

The antibody fragment starting material may be obtained from any speciesincluding for example mouse, rat, rabbit, hamster, camel, llama, goat orhuman. Parts of the antibody fragment may be obtained from more than onespecies, for example the antibody fragments may be chimeric. In oneexample, the constant regions are from one species and the variableregions from another. The antibody fragment starting material may alsobe modified. In another example, the variable region of the antibodyfragment has been created using recombinant DNA engineering techniques.Such engineered versions include those created for example from naturalantibody variable regions by insertions, deletions or changes in or tothe amino acid sequences of the natural antibodies. Particular examplesof this type include those engineered variable region domains containingat least one CDR and, optionally, one or more framework amino acids fromone antibody and the remainder of the variable region domain from asecond antibody. The methods for creating and manufacturing theseantibody fragments are well known in the art (see for example, Boss etal., U.S. Pat. No. 4,816,397; Cabilly et al., U.S. Pat. No. 6,331,415;Shrader et al., WO 92/02551; Ward et al., 1989, Nature, 341, 544;Orlandi et al., 1989, Proc. Natl. Acad. Sci. USA, 86, 3833; Riechmann etal., 1988, Nature, 322, 323; Bird et al, 1988, Science, 242, 423; Queenet al., U.S. Pat. No. 5,585,089; Adair, WO91/09967; Mountain and Adair,1992, Biotechnol. Genet. Eng. Rev, 10, 1-142; Verma et al., 1998,Journal of Immunological Methods, 216, 165-181).

In one embodiment the variable domain pair forming a binding domain is acognate pair. Cognate pair as employed herein is intended to refer to anatural pair of variable domains, that is to say isolated from a singleantibody or antibody expressing cell.

In one example the cognate pair is a complementary V_(H)/V_(L) pairwhich binds the antigen co-operatively i.e. they are a complementaryV_(H)/V_(L) pair.

Typically the cognate pair will be a V_(H)/V_(L) pair derived from thesame antibody.

In one example the cognate pair are a pair of variable domains isolatedas a pair from a ‘library of pairs’, such as a Fab phage displaylibrary.

In one example the V_(H)/V_(L) pair are monospecific.

Variable domains may have been optimized and/or humanized.

Optimised/humanized variable domains derived from a cognate pair willstill be considered a cognate pair after optimization/humanization.

Thus the invention extends to human, humanized or chimeric molecules.

In one embodiment a disulfide bond between a VH and VL pair maycorrespond to the pairs of positions between (unless the contextindicates otherwise Kabat numbering is employed in the list below).Wherever reference is made to Kabat numbering the relevant reference isKabat et al., 1987, in Sequences of Proteins of Immunological Interest,US Department of Health and Human Services, NIH, USA:

-   -   V_(H)37+V_(L)95C see for example Protein Science 6, 781-788 Zhu        et at (1997);    -   V_(H)44+V_(L)100 see for example; Biochemistry 33 5451-5459        Reiter et al (1994); or Journal of Biological Chemistry Vol. 269        No. 28 pp. 18327-18331 Reiter et at (1994); or Protein        Engineering, vol. 10 no. 12 pp. 1453-1459 Rajagopal et at        (1997);    -   V_(H)44+V_(L)105 see for example J Biochem. 118, 825-831 Luo et        at (1995);    -   V_(H)45+V_(L)87 see for example Protein Science 6, 781-788 Zhu        et at (1997);    -   V_(H)55+V_(L)101 see for example FEBS Letters 377 135-139 Young        et at (1995);    -   V_(H)100+V_(L)50 see for example Biochemistry 29 1362-1367        Glockshuber et at (1990);    -   V_(H)100 b+V_(L)49;    -   V_(H)98+V_(L)46 see for example Protein Science 6, 781-788 Zhu        et at (1997);    -   V_(H)101+V_(L)46;    -   V_(H)105+V_(L)43 see for example; Proc. Natl. Acad. Sci. USA        Vol. 90 pp. 7538-7542 Brinkmann et at (1993); or Proteins 19,        35-47 Jung et al (1994); or    -   V_(H)106+V_(L)57 see for example FEBS Letters 377 135-139 Young        et al (1995).

The amino acid pairs listed above are in the positions conducive toreplacement by cysteines such that a disulfide bond can be formed.Cysteines can be engineered into these desired positions by knowntechniques. In one embodiment therefore an engineered cysteine accordingto the present invention refers to where the naturally occurring residueat a given amino acid position has been replaced with a cysteineresidue.

Introduction of engineered cysteines can be performed using any methodknown in the art. These methods include, but are not limited to, PCRextension overlap mutagenesis, site-directed mutagenesis or cassettemutagenesis (see, generally, Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbour Laboratory Press, Cold SpringHarbour, N.Y., 1989; Ausbel et al., Current Protocols in MolecularBiology, Greene Publishing & Wiley-Interscience, NY, 1993).Site-directed mutagenesis kits are commercially available, e.g.QuikChange® Site-Directed Mutagenesis kit (Stratagen, La Jolla, Calif.).Cassette mutagenesis can be performed based on Wells et al., 1985, Gene,34:315-323. Alternatively, mutants can be made by total gene synthesisby annealing, ligation and PCR amplification and cloning of overlappingoligonucleotides.

Accordingly in one embodiment a variable domain pair (V_(H)/V_(L)) ofthe present invention may be linked by a disulfide bond between twocysteine residues, one in V_(H) and one in V_(L), wherein the positionof the pair of cysteine residues is selected from the group consistingof V_(H)37 and V_(L)95, V_(H)44 and V_(L)100, V_(H)44 and V_(L)105,V_(H)45 and V_(L)87, V_(H)100 and V_(L)50, V_(H)100 b and V_(L)49,V_(H)98 and V_(L)46, V_(H)101 and V_(L)46, V_(H)105 and V_(L)43 andV_(H)106 and V_(L)57.

In one embodiment a variable domain pair (V_(H)/V_(L)) of the presentinvention may be linked by a disulfide bond between two cysteineresidues, one in V_(H) and one in V_(L), which are outside of the CDRswherein the position of the pair of cysteine residues is selected fromthe group consisting of V_(H)37 and V_(L)95, V_(H)44 and V_(L)100,V_(H)44 and V_(L)105, V_(H)45 and V_(L)87, V_(H)100 and V_(L)50, V_(H)98and V_(L)46, V_(H)105 and V_(L)43 and V_(H)106 and V_(L)57.

In one embodiment a variable domain pair (V_(H)/V_(L)) of the presentinvention may be linked by a disulfide bond between two cysteineresidues, one in V_(H) and one in V_(L), which are outside of the CDRswherein the position of the pair of cysteine residues is selected fromthe group consisting of V_(H)37 and V_(L)95, V_(H)44 and V_(L)105,V_(H)45 and V_(L)87, V_(H)100 and V_(L)50, V_(H)98 and V_(L)46, V_(H)105and V_(L)43 and V_(H)106 and V_(L)57.

In one embodiment a variable domain pair (V_(H)/V_(L)) of the presentinvention may be linked by a disulfide bond between two cysteineresidues wherein the cysteine residue of V_(H) is at position 44 and thecysteine residue of V_(L) is at position 100.

Typically the cysteine pairs are engineered into those positions inV_(H) and V_(L), accordingly in one embodiment a variable domain pair(V_(H)/V_(L)) of the present invention may be linked by a disulfide bondbetween two engineered cysteine residues, one in V_(H) and one in V_(L),wherein the position of the pair of engineered cysteine residues isselected from the group consisting of V_(H)37 and V_(L)95, V_(H)44 andV_(L)100, V_(H)44 and V_(L)105, V_(H)45 and V_(L)87, V_(H)100 andV_(L)50, V_(H)100 b and V_(L)49, V_(H)98 and V_(L)46, V_(H)101 andV_(L)46, V_(H)105 and V_(L)43 and V_(H)106 and V_(L)57.

In one embodiment a variable domain pair (V_(H)/V_(L)) of the presentinvention may be linked by a disulfide bond between two engineeredcysteine residues, one in VH and one in VL, which are outside of theCDRs wherein the position of the pair of engineered cysteine residues isselected from the group consisting of V_(H)37 and V_(L)95, V_(H)44 andV_(L)100, V_(H)44 and V_(L)105, V_(H)45 and V_(L)87, V_(H)100 andV_(L)50, V_(H)98 and V_(L)46, V_(H)105 and V_(L)43 and V_(H)106 andV_(L)57.

In one embodiment the variable domain pair (V_(H)/V_(L)) is linked by adisulfide bond between two engineered cysteine residues, one in V_(H)and one in V_(L), which are outside of the CDRs wherein the position ofthe pair of engineered cysteine residues is selected from the groupconsisting of V_(H)37 and V_(L)95, V_(H)44 and V_(L)105, V_(H)45 andV_(L)87, V_(H)100 and V_(L)50, V_(H)98 and V_(L)46, V_(H)105 and V_(L)43and V_(H)106 and V_(L)57.

In one embodiment the variable domain pair (V_(H)/V_(L)) is linked by adisulfide bond between two engineered cysteine residues wherein theengineered cysteine residue of V_(H) is at position 44 and theengineered cysteine residue of V_(L) is at position 100.

In one embodiment the molecule specifically binds a target antigen.Specifically binds as employed herein is intended to refer to moleculeshaving high affinity for a target antigen (to which it is specific) andwhich binds antigens to which it is not specific with a low or muchlower affinity (or not at all). Methods of measuring affinity are knownto those skilled in the art and include such assays as BIAcore.

The antibody molecules of the present invention suitably have a highbinding affinity, in particular, nanomolar or picomolar. Affinity may bemeasured using any suitable method known in the art, including BIAcore.In one embodiment the molecule of the present invention has a bindingaffinity of about 100 pM or better. In one embodiment the molecule ofthe present invention has a binding affinity of about 50 pM or better.In one embodiment the molecule of the present invention has a bindingaffinity of about 40 pM or better. In one embodiment the molecule of thepresent invention has a binding affinity of about 30 pM or better. Inone embodiment the molecule of the present invention is fully human orhumanised and has a binding affinity of about 100 pM or better.

In one embodiment there is provided a dimer of Fab′ according to thepresent disclosure for example dimerisation may be through the hinge.

As is well known in the art, a typical Fab molecule comprises a heavyand a light chain pair in which the heavy chain comprises a variableregion V_(H) and a constant domain CH₁ and the light chain comprises avariable region V_(L) and a constant domain CL.

In one embodiment the light chain(s) of the Fab comprises a singleconstant domain CL, for example a natural constant region derived from alight chain, e.g. kappa or lambda.

In one embodiment the heavy chain of the Fab fragment comprises a singleconstant domain, for example a natural or modified CH₁ domain.

Constant domain as employed herein is intended to refer to CH₁, CH₂, CH₃or a constant domain from a light chain. The molecules of the inventionmay include Fc regions i.e —CH₂CH₃ domains.

A derivative of a naturally occurring domain as employed herein isintended to refer to where one, two, three, four or five amino acids ina naturally occurring sequence have been replaced or deleted, forexample to optimize the properties of the domain such as by eliminatingundesirable properties but wherein the characterizing feature(s) of thedomain is/are retained.

In one embodiment a “natural” disulfide bond is present between theconstant domain in the heavy and light chain, for example a disulfidebond, the interchain disulphide bond between CH₁ and CL is present. TheCL domain is derived from either Kappa or Lambda. The natural positionfor a bond forming cysteine in the latter is 214 in human cKappa andcLambda (Kabat numbering 4^(th) edition 1987). A disulfide bond orbond(s) in the constant region of the molecule is/are in addition to theat least one sulfide bond between a variable domain pair.

The exact location of the disulfide-bond-forming cysteine in the heavychain constant domain such as CH₁ depends on the particular domainactually employed. Thus, for example in human gamma-1 the naturalposition of the disulfide bond is located position 233 (Kabat numbering4^(th) edition 1987). The position of the bond forming cysteine forother human isotypes such as gamma 2, 3, 4, IgM and IgD are know, forexample 127.

In one embodiment the molecules according to the disclosure have adisulfide bond in a position equivalent or corresponding to thatnaturally occurring between the heavy chain constant region and thelight chain constant region. Cysteines forming the bonds can beengineered into the domains as required.

In one embodiment a disulfide bond is present between the constantdomain of the heavy and light chain in a non-naturally occurringposition. This may be engineered into the molecule by introducingcysteine(s) into the amino acid chain at the positions required. Thisnon-natural disulfide bond is in addition to or as an alternative to thenatural disulfide bond present between the heavy and light chainconstant domains.

In one embodiment no disulfide bond is present between the heavy andlight chain constant domains, for example one or more of the cysteineresidues may be replaced by another amino acid such as serine.

In one or more embodiments of Fab′ molecules there are no interchaindisulfide bonds in the hinge region.

Alternatively in one or more embodiments one or more (such as two)disulfide bonds are present in the hinge region, for example to allowformation of a F(ab′)₂ or a complete antibody molecule.

In one embodiment a Fab′ or F(ab′)₂ molecule according to the presentdisclosure comprises a modified hinge.

A number of modified hinge regions have already been described forexample, in U.S. Pat. No. 5,677,425, U.S. Pat. No. 6,642,356, WO9915549,WO2005003170, WO2005003169, WO2005003170, WO9825971 and WO2005003171 andthese are incorporated herein by reference. The hinge will usually belocated between the second variable domain in the heavy chain and the Fcregion, where present. Particular examples of hinges include those shownin Table 1.

TABLE 1 Hinge sequences SEQ ID NO: SEQUENCE 1 DKTHTCAA 2 DKTHTCPPCPA 3DKTHTCPPCPATCPPCPA 4 DKTHTCPPCPATCPPCPATCPPCPA 5DKTHTCPPCPAGKPTLYNSLVMSDTAGTCY 6 DKTHTCPPCPAGKPTHVNVSVVMAEVDGTCY 7DKTHTCCVECPPCPA 8 DKTHTCPRCPEPKSCDTPPPCPRCPA 9 DKTHTCPSCPA

The inventors believe that by providing variable domains as cognatepairs in the final construct optimizes and maintains the desirableantigen binding properties of the binding site formed by the relevantpair.

In one embodiment the antibody molecules of the present invention, inparticular, Fab or Fab′ molecules of the present invention comprise oneor more albumin binding peptides. In vivo the peptide binds albumin,which increases the half-life of the molecule.

The albumin binding peptide may be appended from one or more variableregions, a hinge or C-terminal of the molecule or any location that doesnot interfere with the molecules antigen binding properties.

Examples of albumin binding peptides are provided in WO 2007/106120 andinclude:

TABLE 2 SEQ ID NO: SEQUENCE 10 DLCLRDWGCLW 11 DICLPRWGCLW 12MEDICLPRWGCLWGD 13 QRLMEDICLPRWGCLWEDDE 14 QGLIGDICLPRWGCLWGRSV 15QGLIGDICLPRWGCLWGRSVK 16 EDICLPRWGCLWEDD 17 RLMEDICLPRWGCLWEDD 18MEDICLPRWGCLWEDD 19 MEDICLPRWGCLWED 20 RLMEDICLARWGCLWEDD 21EVRSFCTRWPAEKSCKPLRG 22 RAPESFVCYWETICFERSEQ 23 EMCYFPGICWM

It will be appreciated that one or more amino acid substitutions,additions and/or deletions may be made to the variable domains, providedby the present invention, without significantly altering the ability ofthe antibody molecule, in particular a Fab or Fab′ to bind to targetantigen and to neutralise activity thereof. The effect of any amino acidsubstitutions, additions and/or deletions can be readily tested by oneskilled in the art, for example by using the in vitro assays, forexample a BIAcore assay.

The constant region domains, in particular in the Fc domain, wherepresent, employed in the present invention, may be selected havingregard to the proposed function of the molecule, in particular theeffector functions which may be required, and for example, may be humanIgA, IgD, IgE, IgG or IgM domains. In particular, domains from human IgGmay be used, especially of the IgG1 and IgG3 isotypes when the Fab orFab′ is intended for therapeutic uses and antibody effector functionsare required. Alternatively, domains from IgG2 and IgG4 isotypes may beused when the Fab or Fab′ is intended for therapeutic purposes andantibody effector functions are not required. It will be appreciatedthat sequence variants of these constant region domains may also beused. For example domains from IgG4 molecules in which the serine atposition 241 has been changed to proline as described in Angal et al.,Molecular Immunology, 1993, 30 (1), 105-108 may be used. It will also beunderstood by one skilled in the art that Fab or Fab′ may undergo avariety of posttranslational modifications. The type and extent of thesemodifications often depends on the host cell line used to express themolecule as well as the culture conditions. Such modifications mayinclude variations in glycosylation, methionine oxidation,diketopiperazine formation, aspartate isomerization and asparaginedeamidation. A frequent modification is the loss of a carboxy-terminalbasic residue (such as lysine or arginine) due to the action ofcarboxypeptidases (as described in Harris, R J. Journal ofChromatography 705:129-134, 1995).

If desired a molecule for use in the present invention may be conjugatedto one or more effector molecule(s). It will be appreciated that theeffector molecule may comprise a single effector molecule or two or moresuch molecules so linked as to form a single moiety that can be attachedto the antibody molecule in particular, Fab or Fab′ of the presentinvention. Where it is desired to obtain a fragment according to theinvention linked to an effector molecule, this may be prepared bystandard chemical or recombinant DNA procedures in which the fragment islinked either directly or via a coupling agent to the effector molecule.Techniques for conjugating such effector molecules to Fab/Fab′ are wellknown in the art (see, Hellstrom et al., Controlled Drug Delivery, 2ndEd., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982,Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology andTherapeutics, 83, 67-123). Particular chemical procedures include, forexample, those described in WO 93/06231, WO 92/22583, WO 89/00195, WO89/01476 and WO03031581. Alternatively, where the effector molecule is aprotein or polypeptide the linkage may be achieved using recombinant DNAprocedures, for example as described in WO 86/01533 and EP0392745.

The term effector molecule as used herein includes, for example,antineoplastic agents, drugs, toxins, biologically active proteins, forexample enzymes, other antibody or antibody fragments, synthetic ornaturally occurring polymers, nucleic acids and fragments thereof e.g.DNA, RNA and fragments thereof, radionuclides, particularly radioiodide,radioisotopes, chelated metals, nanoparticles and reporter groups suchas fluorescent compounds or compounds which may be detected by NMR orESR spectroscopy.

Examples of effector molecules may include cytotoxins or cytotoxicagents including any agent that is detrimental to (e.g. kills) cells.Examples include combrestatins, dolastatins, epothilones, staurosporin,maytansinoids, spongistatins, rhizoxin, halichondrins, roridins,hemiasterlins, taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof.

Effector molecules also include, but are not limited to, antimetabolites(e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g. daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g. dactinomycin (formerly actinomycin),bleomycin, mithramycin, anthramycin (AMC), calicheamicins orduocarmycins), and anti-mitotic agents (e.g. vincristine andvinblastine).

Other effector molecules may include chelated radionuclides such as¹¹¹In and ⁹⁰Y, Lu¹⁷⁷, Bismuth²¹³, Californium²⁵², Iridium¹⁹² andTungsten¹⁸⁸/Rhenium¹⁸⁸; or drugs such as but not limited to,alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

Other effector molecules include proteins, peptides and enzymes. Enzymesof interest include, but are not limited to, proteolytic enzymes,hydrolases, lyases, isomerases, transferases. Proteins, polypeptides andpeptides of interest include, but are not limited to, immunoglobulins,toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheriatoxin, a protein such as insulin, tumour necrosis factor, α-interferon,β-interferon, nerve growth factor, platelet derived growth factor ortissue plasminogen activator, a thrombotic agent or an anti-angiogenicagent, e.g. angiostatin or endostatin, or, a biological responsemodifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2(IL-2), granulocyte macrophage colony stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF)or other growth factor and immunoglobulins.

Other effector molecules may include detectable substances useful forexample in diagnosis. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive nuclides, positronemitting metals (for use in positron emission tomography), andnonradioactive paramagnetic metal ions. See generally U.S. Pat. No.4,741,900 for metal ions which can be conjugated to antibodies for useas diagnostics. Suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;suitable prosthetic groups include streptavidin, avidin and biotin;suitable fluorescent materials include umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride and phycoerythrin; suitable luminescentmaterials include luminol; suitable bioluminescent materials includeluciferase, luciferin, and aequorin; and suitable radioactive nuclidesinclude ¹²⁵I, ¹³¹I, ¹¹¹In and ⁹⁹Tc.

In another example the effector molecule may increase the half-life ofthe Fab/Fab′ in vivo, and/or reduce immunogenicity of the antibodyand/or enhance the delivery of an antibody across an epithelial barrierto the immune system. Examples of suitable effector molecules of thistype include polymers, albumin, albumin binding proteins or albuminbinding compounds such as those described in WO 05/117984.

Where the effector molecule is a polymer it may, in general, be asynthetic or a naturally occurring polymer, for example an optionallysubstituted straight or branched chain polyalkylene, polyalkenylene orpolyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g.a homo- or hetero-polysaccharide.

Specific optional substituents which may be present on theabove-mentioned synthetic polymers include one or more hydroxy, methylor methoxy groups.

Specific examples of synthetic polymers include optionally substitutedstraight or branched chain poly(ethyleneglycol),poly(propyleneglycol)poly(vinylalcohol) or derivatives thereof,especially optionally substituted poly(ethyleneglycol) such asmethoxypoly(ethyleneglycol) or derivatives thereof.

Specific naturally occurring polymers include lactose, amylose, dextran,glycogen or derivatives thereof.

“Derivatives” as used herein is intended to include reactivederivatives, for example thiol-selective reactive groups such asmaleimides and the like. The reactive group may be linked directly orthrough a linker segment to the polymer. It will be appreciated that theresidue of such a group will in some instances form part of the productas the linking group between the antibody fragment of the disclosure andthe polymer.

The size of the polymer may be varied as desired, but will generally bein an average molecular weight range from 500 Da to 50000 Da, forexample from 5000 to 40000 Da such as from 20000 to 40000 Da. Thepolymer size may in particular be selected on the basis of the intendeduse of the product for example ability to localize to certain tissuessuch as tumors or extend circulating half-life (for review see Chapman,2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example,where the product is intended to leave the circulation and penetratetissue, for example for use in the treatment of a tumour, it may beadvantageous to use a small molecular weight polymer, for example with amolecular weight of around 5000 Da. For applications where the productremains in the circulation, it may be advantageous to use a highermolecular weight polymer, for example having a molecular weight in therange from 20000 Da to 40000 Da.

Suitable polymers include a polyalkylene polymer, such as apoly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) or aderivative thereof, and especially with a molecular weight in the rangefrom about 15000 Da to about 40000 Da.

In one example a Fab/Fab′ for use in the present invention are attachedto poly(ethyleneglycol) (PEG) moieties. In one particular example theFab/Fab′ is an antibody fragment and the PEG molecules may be attachedthrough any available amino acid side-chain or terminal amino acidfunctional group located in the antibody fragment, for example any freeamino, imino, thiol, hydroxyl or carboxyl group. Such amino acids mayoccur naturally in the antibody fragment or may be engineered into thefragment using recombinant DNA methods (see for example U.S. Pat. No.5,219,996; U.S. Pat. No. 5,667,425; WO 98/25971). In one example themolecule of the present invention is a modified Fab fragment wherein themodification is the addition to the C-terminal end of its heavy chainone or more amino acids to allow the attachment of an effector molecule.Suitably, the additional amino acids form a modified hinge regioncontaining one or more cysteine residues to which the effector moleculemay be attached. Multiple sites can be used to attach two or more PEGmolecules.

In one embodiment a PEG molecule is linked to a cysteine 171 in thelight chain, for example see WO2008/038024 incorporated herein byreference.

Suitably PEG molecules are covalently linked through a thiol group of atleast one cysteine residue located in the antibody fragment. Eachpolymer molecule attached to the modified antibody fragment may becovalently linked to the sulphur atom of a cysteine residue located inthe fragment. The covalent linkage will generally be a disulphide bondor, in particular, a sulphur-carbon bond. Where a thiol group is used asthe point of attachment appropriately activated effector molecules, forexample thiol selective derivatives such as maleimides and cysteinederivatives may be used. An activated polymer may be used as thestarting material in the preparation of polymer-modified antibodyfragments as described above. The activated polymer may be any polymercontaining a thiol reactive group such as an α-halocarboxylic acid orester, e.g. iodoacetamide, an imide, e.g. maleimide, a vinyl sulphone ora disulphide. Such starting materials may be obtained commercially (forexample from Nektar, formerly Shearwater Polymers Inc., Huntsville,Ala., USA) or may be prepared from commercially available startingmaterials using conventional chemical procedures. Particular PEGmolecules include 20K methoxy-PEG-amine (obtainable from Nektar,formerly Shearwater; Rapp Polymere; and SunBio) and M-PEG-SPA(obtainable from Nektar, formerly Shearwater).

The present invention also provides isolated DNA encoding an antibodymolecule described herein or a fragment thereof, e.g. Fab or Fab′.

In a further aspect there is provided a vector comprising said DNA.

General methods by which the vectors may be constructed, transfectionmethods and culture methods are well known to those skilled in the art.In this respect, reference is made to “Current Protocols in MolecularBiology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and theManiatis Manual produced by Cold Spring Harbor Publishing.

Molecule or fragment according to the present invention, are usedinterchangeably herein.

In a further aspect there is provided a host cell comprising said vectorand/or DNA.

Any suitable host cell/vector system may be used for expression of theDNA sequences encoding the molecule of the present invention. Bacterial,for example E. coli, and other microbial systems may be used oreukaryotic, for example mammalian, host cell expression systems may alsobe used. Suitable mammalian host cells include CHO, myeloma or hybridomacells.

The present invention also provides a process for the production of anantibody molecule as described herein comprising culturing a host cellcontaining a vector (and/or DNA) of the present invention underconditions suitable for leading to expression of protein from DNAencoding an antibody molecule of the present invention, and isolating anantibody molecule such as a Fab or Fab′.

For production of products comprising both heavy and light chains, thecell line may be transfected with two vectors, a first vector encoding alight chain polypeptide and a second vector encoding a heavy chainpolypeptide. Alternatively, a single vector may be used, the vectorincluding sequences encoding light chain and heavy chain polypeptides.

The antibody molecules according to the present disclosure are expressedat suitable levels from host cells making them conducive to commercialprocessing.

The antibody molecules of the present invention are useful in thetreatment and/or prophylaxis of a pathological condition.

Thus there is provided an antibody or antibody fragment comprising a Fabmolecule according to the present invention for use in treatment, byadministering a therapeutically effective amount thereof, for example ina pharmaceutical formulation. In one embodiment the antibody or antibodyfragment according to the invention is administered topically to thelungs, for example by inhalation.

The present invention also provides a pharmaceutical or diagnosticcomposition comprising a molecule of the present invention incombination with one or more of a pharmaceutically acceptable excipient,diluent or carrier. Accordingly, provided is the use of a molecule ofthe invention for the manufacture of a medicament. The composition willusually be supplied as part of a sterile, pharmaceutical compositionthat will normally include a pharmaceutically acceptable carrier. Apharmaceutical composition of the present invention may additionallycomprise a pharmaceutically-acceptable adjuvant.

The present invention also provides a process for preparation of apharmaceutical or diagnostic composition comprising adding and mixingthe molecule of the present invention together with one or more of apharmaceutically acceptable excipient, diluent or carrier.

The molecule of the disclosure may be the sole active ingredient in thepharmaceutical or diagnostic composition or may be accompanied by otheractive ingredients including other antibody ingredients, for exampleanti-TNF, anti-IL-1β, anti-T cell, anti-IFNγ or anti-LPS antibodies, ornon-antibody ingredients such as xanthines. Other suitable activeingredients include antibodies capable of inducing tolerance, forexample, anti-CD3 or anti-CD4 antibodies.

In a further embodiment the molecule or composition according to thedisclosure is employed in combination with a further pharmaceuticallyactive agent, for example a corticosteroid (such as fluticasonoepropionate) and/or a beta-2-agonist (such as salbutamol, salmeterol orformoterol) or inhibitors of cell growth and proliferation (such asrapamycin, cyclophosphmide, methotrexate) or alternative a CD28 and/orCD40 inhibitor. In one embodiment the inhitor is a small molecule. Inanother embodiment the inhibitor is an antibody specific to the target.

The pharmaceutical compositions suitably comprise a therapeuticallyeffective amount of the molecule of the invention. The term“therapeutically effective amount” as used herein refers to an amount ofa therapeutic agent needed to treat, ameliorate or prevent a targeteddisease or condition, or to exhibit a detectable therapeutic orpreventative effect. The therapeutically effective amount can beestimated initially either in cell culture assays or in animal models,usually in rodents, rabbits, dogs, pigs or primates. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

The precise therapeutically effective amount for a human subject willdepend upon the severity of the disease state, the general health of thesubject, the age, weight and gender of the subject, diet, time andfrequency of administration, drug combination(s), reaction sensitivitiesand tolerance/response to therapy. This amount can be determined byroutine experimentation and is within the judgment of the clinician.Generally, a therapeutically effective amount will be from 0.01 mg/kg to50 mg/kg, for example 0.1 mg/kg to 20 mg/kg. Pharmaceutical compositionsmay be conveniently presented in unit dose forms containing apredetermined amount of an active agent of the invention per dose.

Compositions may be administered individually to a patient or may beadministered in combination (e.g. simultaneously, sequentially orseparately) with other agents, drugs or hormones.

The dose at which a molecule of the present invention is administereddepends on the nature of the condition to be treated, for example theextent of the disease/inflammation present and on whether the moleculeis being used prophylactically or to treat an existing condition.

The frequency of dose will depend on the half-life of the molecule andthe duration of its effect. If the molecule has a short half-life (e.g.2 to 10 hours) it may be necessary to give one or more doses per day.Alternatively, if the molecule has a long half life (e.g. 2 to 15 days)it may only be necessary to give a dosage once per day, once per week oreven once every 1 or 2 months.

The pharmaceutically acceptable carrier should not itself induce theproduction of antibodies harmful to the individual receiving thecomposition and should not be toxic. Suitable carriers may be large,slowly metabolised macromolecules such as proteins, polypeptides,liposomes, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers and inactive virusparticles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the patient.

Suitable forms for administration include forms suitable for parenteraladministration, e.g. by injection or infusion, for example by bolusinjection or continuous infusion. Where the product is for injection orinfusion, it may take the form of a suspension, solution or emulsion inan oily or aqueous vehicle and it may contain formulatory agents, suchas suspending, preservative, stabilising and/or dispersing agents.Alternatively, the molecule of the disclosure may be in dry form, forreconstitution before use with an appropriate sterile liquid.

Once formulated, the compositions of the invention can be administereddirectly to the subject. The subjects to be treated can be animals.However, in one or more embodiments the compositions are adapted foradministration to human subjects.

Suitably in formulations according to the present disclosure, the pH ofthe final formulation is not similar to the value of the isoelectricpoint of the molecule or fragment, for example if the pH of theformulation is 7 then a pI of from 8-9 or above may be appropriate.Whilst not wishing to be bound by theory it is thought that this mayultimately provide a final formulation with improved stability, forexample the binding protein or fragment thereof remains in solution.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, transcutaneous (for example, seeWO98/20734), subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, intravaginal or rectal routes. Hyposprays may alsobe used to administer the pharmaceutical compositions of the invention.Typically, the therapeutic compositions may be prepared as injectables,either as liquid solutions or suspensions. Solid forms (for examplelyophilised forms) suitable for solution in, or suspension in, liquidvehicles prior to injection may also be prepared.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Dosage treatmentmay be a single dose schedule or a multiple dose schedule.

It will be appreciated that the active ingredient in the compositionwill be a molecule. As such, it will be susceptible to degradation inthe gastrointestinal tract. Thus, if the composition is to beadministered by a route using the gastrointestinal tract, thecomposition will need to contain agents which protect the bindingprotein from degradation but which release the molecule once it has beenabsorbed from the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Remington's Pharmaceutical Sciences (Mack PublishingCompany, N.J. 1991).

In one embodiment the formulation is provided as a formulation fortopical administrations including inhalation.

Suitable inhalable preparations include inhalable powders, meteringaerosols containing propellant gases or inhalable solutions free frompropellant gases. Inhalable powders according to the disclosurecontaining the active substance may consist solely of the abovementionedactive substances or of a mixture of the abovementioned activesubstances with physiologically acceptable excipient.

These inhalable powders may include monosaccharides (e.g. glucose orarabinose), disaccharides (e.g. lactose, saccharose, maltose), oligo-and polysaccharides (e.g. dextranes), polyalcohols (e.g. sorbitol,mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) ormixtures of these with one another. Mono- or disaccharides are suitablyused, the use of lactose or glucose, particularly but not exclusively inthe form of their hydrates.

Particles for deposition in the lung require a particle size less than10 microns, such as 1-9 microns for example from 0.1 to 5 μm, inparticular from 1 to 5 μm. The particle size of the active ingredient(such as the binding protein or fragment) is of primary importance.

The propellent gases which can be used to prepare the inhalable aerosolsare known in the art. Suitable propellent gases are selected from amonghydrocarbons such as n-propane, n-butane or isobutane andhalohydrocarbons such as chlorinated and/or fluorinated derivatives ofmethane, ethane, propane, butane, cyclopropane or cyclobutane. Theabovementioned propellent gases may be used on their own or in mixturesthereof.

Particularly suitable propellent gases are halogenated alkanederivatives selected from among TG 11, TG 12, TG 134a and TG227. Of theabovementioned halogenated hydrocarbons, TG134a(1,1,1,2-tetrafluoroethane) and TG227 (1,1,1,2,3,3,3-heptafluoropropane)and mixtures thereof are particularly suitable.

The propellent-gas-containing inhalable aerosols may also contain otheringredients such as cosolvents, stabilisers, surface-active agents(surfactants), antioxidants, lubricants and means for adjusting the pH.All these ingredients are known in the art.

The propellant-gas-containing inhalable aerosols according to theinvention may contain up to 5% by weight of active substance. Aerosolsaccording to the invention contain, for example, 0.002 to 5% by weight,0.01 to 3% by weight, 0.015 to 2% by weight, 0.1 to 2% by weight, 0.5 to2% by weight or 0.5 to 1% by weight of active ingredient.

Alternatively topical administrations to the lung may also be byadministration of a liquid solution or suspension formulation, forexample employing a device such as a nebulizer, for example, a nebulizerconnected to a compressor (e.g., the Pari LC-Jet Plus® nebulizerconnected to a Pari Master® compressor manufactured by Pari RespiratoryEquipment, Inc., Richmond, Va.).

The molecule of the invention can be delivered dispersed in a solvent,e.g., in the form of a solution or a suspension. It can be suspended inan appropriate physiological solution, e.g., saline or otherpharmacologically acceptable solvent or a buffered solution. Bufferedsolutions known in the art may contain 0.05 mg to 0.15 mg disodiumedetate, 8.0 mg to 9.0 mg NaCl, 0.15 mg to 0.25 mg polysorbate, 0.25 mgto 0.30 mg anhydrous citric acid, and 0.45 mg to 0.55 mg sodium citrateper 1 mL of water so as to achieve a pH of about 4.0 to 5.0. Asuspension or solution can be reconstituted, for example, lyophilisedmolecules.

The therapeutic suspensions or solution formulations can also containone or more excipients. Excipients are well known in the art and includebuffers (e.g., citrate buffer, phosphate buffer, acetate buffer andbicarbonate buffer), amino acids, urea, alcohols, ascorbic acid,phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride,liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensionscan be encapsulated in liposomes or biodegradable microspheres. Theformulation will generally be provided in a substantially sterile formemploying sterile manufacture processes.

This may include production and sterilization by filtration of thebuffered solvent/solution used for the formulation, aseptic suspensionof the molecule in the sterile buffered solvent solution, and dispensingof the formulation into sterile receptacles by methods familiar to thoseof ordinary skill in the art.

Nebulizable formulation according to the present disclosure may beprovided, for example, as single dose units (e.g., sealed plasticcontainers or vials) for example packed in foil envelopes. Each vialcontains a unit dose in a volume, e.g., 2 mL, of solvent/solutionbuffer.

The molecules of the present disclosure are thought to be particularlysuitable for delivery via nebulisation.

Comprising in the context of the present specification is intended tomeaning including.

Where technically appropriate embodiments of the invention may becombined.

Embodiments are described herein as comprising certainfeatures/elements. The disclosure also extends to separate embodimentsconsisting or consisting essentially of said features/elements.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of the embodiments of the presentinvention can be best understood when read in conjunction with thefollowing drawings, where like structure is indicated with likereference numerals and in which:

FIG. 1 shows the sequences for 497Fab-His (heavy chain—SEQ ID NO:24;light chain—SEQ ID NO:25); 497dsFab-His (heavy chain—SEQ ID NO:26; lightchain—SEQ ID NO:27); 652Fab-His (heavy chain—SEQ ID NO:28; lightchain—SEQ ID NO:29); and 652dsFab-His (heavy chain—SEQ ID NO:30; lightchain—SEQ ID NO:31).

EXAMPLE 1 Generation and Analysis of Disulphide Stabilized Fab-his

Construction of 652Fab-His, 652dsFab-His, 497Fab-His and 497dsFab-HisPlasmids

The light chain variable regions of 652 and 497 antibodies were clonedas HindIII/BsiW1 fragment into UCB's proprietary mammalian expressionCKappa vector. The heavy chain variable regions of 652 and 497antibodies were cloned as HindIII-Xho1 fragment into CH₁-His vector.

Disulphide stabilization by mutation to cysteine at position 44 of theheavy chain and 100 of the light chain were done using the QuikChangeLightening Site directed mutagenesis kit from Agilent Technologies,using the manufacturer's protocol. Two 30 base pair oligos were designedfor introducing the cysteine mutation at the heavy 44 and light 100amino acid positions. The disulphide stabilized constructs were verifiedby sequencing. The sequences for the antibody are shown in FIG. 1.

Mammalian Expression of 652Fab-His, 652dsFab-His, 497Fab-His and497dsFab-His

HEK293 cells were transfected with the heavy and light chain plasmidsusing Invitrogen's 293fectin transfection reagent according to themanufacturer's instructions. Briefly, 5 μg heavy chain plasmid and 5 μglight chain plasmid were incubated with 10 μl 293fectin and 340 μlOptimem media for 20 mins at RT. The mixture was then added to 5×10⁶HEK293 cells in suspension and incubated for 4 days with shaking at 37°C. After 4 days the supernatant was collected by centrifugation at1500×g to remove the cells and then 0.22 μm sterile filtered.

652Fab-His, 652dsFab-His, 497Fab-His and 497dsFab-His Quantification

The concentration of Fab or dsFab in the mammalian supernatants may bemeasured using a sandwich ELISA. The Fab or dsFab in the sample iscaptured with an anti-CH₁ antibody and detected with an anti-kappa-HRPconjugate. The detection antibody is then developed with TMB and theconcentration of the unknown samples calculated from a standard curve.

652Fab-His, 652dsFab-His, 497Fab-His and 497dsFab-His Purification

The 652Fab-His, 652dsFab-His, 497Fab-His and 497dsFab-His in thesupernatants were purified by Ni affinity chromatography. To theappropriate number of wells of a Qiagen 96 well filter plate was added150 μl of a 50% slurry of NiNTI superflow and the unused wells werecovered with a plate seal. −0.5 Bar vacuum was applied using a Milliporevacuum manifold. To each well was added 800 μl of 50 mM sodiumphosphate, 300 mM sodium chloride, pH 8 buffer and a −0.5 Bar vacuum wasapplied. To each well was added 800 μl of 250 mM imidazole, 50 mM sodiumphosphate, 300 mM sodium chloride, pH 8 buffer and a −0.5 Bar vacuum wasapplied. To each well was added 800 μl of 50 mM sodium phosphate, 300 mMsodium chloride, pH 8 buffer and a −0.5 Bar vacuum was applied. To eachwell was added 800 μl of 50 mM sodium phosphate, 300 mM sodium chloride,pH 8 buffer and a −0.5 Bar vacuum was applied. To each sterile filteredsupernatant was added a 10^(th) volume of 100 mM imidazole, 0.5M sodiumphosphate, 1.5M sodium chloride, pH 8 buffer. 800 μl of the conditionedsupernatants were applied to the wells, left for 5 minutes and then a−0.5 Bar vacuum was applied. This was repeated until all the conditionsupernatant had been applied. The wells were washed by the addition of800 μl of 20 mM imidazole, 50 mM sodium phosphate, 300 mM sodiumchloride, pH 8 buffer and a −0.5 Bar vacuum was applied. The wash stepwas repeated 3 times. A clean 96 deep well plate was placed under thefilter plate and the Fab-His was eluted into this plate by the additionof 100 μl of 250 mM imidazole, 50 mM sodium phosphate, 300 mM sodiumchloride, pH 8 buffer to the filter plate wells followed by applicationof a −0.5 Bar vacuum.

Thermofluor Thermal Stability Assay

Samples (1 μl of sample at ˜1 mg/ml, 8 μl of PBS and 1 μl of 30× stockof Sypro orange fluorescent dye) may be run in quadruplicate in 384 wellplates. The plate is heated from 20-99° C. using a 7900HT fast real-timePCR system and the fluorescence (excitation at 490 nm, emission at 530nm) measured. The data is processed and the inflection point forunfolding calculated.

1. An antibody or antibody fragment comprising at least one Fabmolecule, wherein the Fab molecule comprises one or more disulfide bondspresent between variable domains of heavy and light chain pairingtherein.
 2. The antibody or antibody fragment of claim 1, wherein theantibody or antibody fragment is selected from the group consisting of aFab, a Fab′, a F(ab)₂ and a complete antibody molecule.
 3. The antibodyor antibody fragment of claim 1, wherein the antibody or antibodyfragment comprises two Fab molecules, wherein in each Fab molecule, oneor more disulfide bonds are present between a light chain variableregion, V_(L) and a heavy chain variable region, V_(H).
 4. The antibodyor antibody fragment of claim 3, wherein the antibody or antibodyfragment is complete antibody molecule or F(ab)₂.
 5. The antibody orantibody fragment of claim 1, wherein the molecule further comprises analbumin binding peptide.
 6. The antibody or antibody fragment of claim1, wherein one disulfide bond is present between the light chainvariable region, V_(L) and the heavy chain variable region, V_(H) of theFab molecule.
 7. The antibody or antibody fragment of claim 1, whereinone disulfide bond is present between each light chain variable region,V_(L) and the heavy chain variable region, V_(H) pairing of the antibodyor fragment.
 8. A composition comprising the antibody or antibodyfragment of claim 1, and a pharmaceutically acceptable excipient.
 9. Theantibody or antibody fragment of claim 1 or a composition comprising anantibody or antibody fragment according to claim 1, and apharmaceutically acceptable excipient for use in treatment orprophylaxis.
 10. A method of treatment comprising administering atherapeutically effective amount of an antibody or fragment as definedin claim 1 or a composition comprising an antibody or fragment accordingto claim 1, and a pharmaceutically acceptable excipient.
 11. Theantibody or antibody fragment of claim 1 or a composition comprising anantibody or antibody fragment according to claim 1 for the manufactureof a medicament.
 12. The antibody or antibody fragment of claim 1,wherein the disulfide bond is between two cysteine residues, one in theV_(L) and one in the V_(H), wherein the cysteine in the V_(L) is atposition 100 and the cysteine in the V_(H) is at position
 44. 13. Anantibody or antibody fragment comprising at least one Fab molecule,wherein the FAB molecule comprises one or more disulfide bonds presentbetween variable domains of heavy and light chain pairing therein, andwherein the molecule further comprises an albumin binding peptide.